Chicken anaemia agent broiler vaccine

ABSTRACT

The present invention for the first time discloses a health problem in broilers at about 3-5 weeks, resulting in significant production losses, and demonstrates that these production losses can be prevented by a live attenuated CAA vaccine for mucosal administration.

This application is a 371 of PCT/EP96/00122 filed Jan. 6, 1996.

The present invention is concerned with a live attenuated CAA vaccine.

Chicken anaemia agent (CAA) is the causative agent of a disease known asavian infectious anaemia, anaemia dermatitis syndrome or blue-wingdisease and was first described by Yuasa et al. in 1979 (Avian Diseases23, 366-385, 1979).

Most outbreaks of naturally occurring CAA-induced disease have beenreported in broilers. The disease is acute and the first signs usuallyoccur at 10-14 days of age. This clinical disease is characterized by asudden increase in mortality, usually around 5-10%, but up to 60% hasbeen reported. Peak mortality occurs within 5 to 6 days of onset ofdisease. Further clinical signs include depression and anorexia.Moreover, severe anaemia, hemorrhages throughout the body, atrophy ofthe thymus and bursa of Fabricius and yellowish bone marrow is seen inaffected chickens McNulty, M. S., Avian Pathol. 20, 187-203, 1991).

The clinical syndrome in broilers occurs in commercial flocks whenin-lay breeders with no previous exposure to the virus, i.e.seronegative breeders, become infected. CAA is transmitted vertically tothe progeny, which develop the disease at 10 to 14 days. No clinicalsigns have been reported in the infected breeders, and there is noapparent effect on egg production, hatchability or fertility, becausechicks develop an age resistance to CAA-induced disease.

The anaemia dermatitis syndrome in chickens caused by the verticalspread of CAA from the breeder through the egg to the progeny can beprevented by ensuring that the parent flocks develop antibodies to CAAbefore the onset of lay. This can occur through natural exposure of theflocks to the virus. However, due to the significant economical lossesresulting from clinical CAA it is preferable to vaccinate the parentstock in order to induce a sero-conversion in breeder flocks during therearing period. Older birds inoculated with the virus developsignificant titers of neutralising antibodies at about 3 weeks afterinfection. These virus neutralizing antibodies are passed from thebreeder to the progeny via the egg such that the chickens obtain a titerof maternal antibody which protects the young chickens against CAAinfection during the first weeks after hatching.

Yuasa, N. et al. (Avian Diseases 24, 197-201, 1980) disclose thatcommercial chickens are generally refractory to CAA field infection andthat this was closely related to the presence of maternal antibodies inthe chick. Otaki, Y. et al. (Avian Pathology 21, 147-151, 1992) showedthat even a very low level of maternal antibody is effective forpreventing CAA-infection. Maternal antibodies could be detected up to 3to 5 weeks of age. In a study using chickens with maternal derivedantibodies (MDA) against CAA Yuasa, N. (Poultry Diseases, Proc. 2ndAsian/Pacific Poultry Health Conference, 385-406, 1988) has shown thatthe antibody-positive rate starts decreasing at about 3 weeks of age.

Vielitz et al. (J. Vet. Medicine 34, 553-557, 1987) describes a CAAvaccine comprising the not-attenuated Cux-1 anaemia pathogen suitablefor vaccinating parent stock at the age of 13-15 weeks. Althoughchickens vaccinated intramuscularly with this vaccine developedantibodies 2 weeks after vaccination, in chickens vaccinated viadrinking water antibodies were observed only from 4 weeks afteradministration of the vaccine.

Recently, the first live attenuated vaccine for administration to olderbirds has been disclosed in European patent application No. 0533294(Akzo Nobel N. V.). Although this vaccine was able to elicit asufficient amount of CAA neutralizing antibodies in these birds whenadministered parentally, vaccination with the live attenuated vaccinevia the drinking water or spray route did not induce an adequate immuneresponse (Steenhuisen, W. et al., International symposium on infectiousbursal disease and chicken infectious anaemia, Rauischholzhausen,Germany, 1994).

In addition to the vertically spreading of CAA in chickens the virusalso spreads horizontally. Horizontal spread in broilers probably occursthrough ingestion of faecally contaminated material or contaminatedfomites. Horizontal acquired infections in broiler progeny of immunebreeder flocks also occur when MDA against CAA are declining. However,the effect of horizontal CAA infection in broilers is not clear.

McNulty et al. (Avian Diseases 35, 263-268, 1991) compared productionand performance parameters of clinically normal broiler flocks which hadantibody to CAA at slaughter with clinically normal broiler flocks whichhad no antibody to CAA at slaughter and reported that subclinical CAA inbroilers of about 6 weeks of age was associated with significanteconomic losses. It was suggested to devise a live attenuated vaccinefor broilers to decrease the losses associated with subclinical CAA.Contrary to this Goodwin, M. A. et al. (Avian Diseases 37, 542-545,1993) found no differences in weight gain or production performanceamong healthy CAA-antibody positive and negative SPF chicks and broilerchicks.

The existence of a new type of health problems in broilers and itssolution is for the first time disclosed herein. This type of problemcan be observed in broilers from about 3 weeks of age and ischaracterized by the following symptoms:

an arrest of growth during the third or fourth week of age

a decrease of the feed-conversion

increased leg problems and lameness

slight increase of mortality towards the end of the growing period

pale appearance

undigested feed in the feaces. These problems resulted in successiveproduction rounds with disappointing performance results in broilerfarms with previous good performance records.

An object of the present invention is to provide a vaccine which is ableto prevent this performance drop syndrome affecting broiler flocksduring the growth period, taking into account that the vaccine must havea beneficial effect in the face of maternal antibodies.

A further object of the present invention is to provide a vaccine whichis able to induce a beneficial response within a very short time afteradministration in order to prevent the early performance drop inbroilers.

The present invention provides a vaccine for mucosal administration forthe protection of broilers against production drop and comprising a liveattenuated CAA virus and a pharmaceutically acceptable carrier.

It is surprising, that a live attenuated CAA vaccine for mucosaladministration is able to stimulate the broiler's immune system within avery short time after administration, which is a prerequisite for aneffective prevention of the production drop syndrome which affects thebroilers already from about 3 weeks of age, because a CAA vaccine forparent stock derived from a virulent CAA, administered via the mucosalroute, i.e. via drinking water developed an immune response only from 4weeks onwards after administration (Vielitz et al., supra). In addition,with respect to the live attenuated CAA vaccine disclosed in EPA0533294, it was demonstrated that such a vaccine in a form suitable forspray or drinking water administration did not induce an adequate immuneresponse (Steenhuisen, W. et al., supra) in older birds.

The term broiler refers to a young, tender chicken, specially producedfor the table. These fast growing chicks specially reared for theslaughter are the progeny of special types (heavy) breeder hens andcocks. For example, female broiler breeders are usually derived of theWhite Plymouth Rock-Sussex or New Hampshire breeds. Broiler breedermales are usually derived from the Cornish breed, such as Cornish Indiangame.

The vaccine according to the present invention is in a form suitable formucosal administration. This means that the vaccine is administered suchthat it is brought into contact with the broiler's mucosal membranes,for example of the respiratory tract, intestinal tract or the eye.Therefore, the vaccine can be applied intranasally, orally,intraocularly or intracloacal.

In a preferred embodiment of the invention the vaccine for intranasal ororal administration is in a form suitable for administration by spray,including aerosol, or drinking water, respectively.

The spray or aerosol method involves the administration of the liveattenuated CAA virus vaccine incorporated in small liquid particles. Inthe former method particles usually have an initial droplet size rangingfrom 10 to 100 microns and in the latter method from <1 to 50 microns.

In order to prevent inactivation of the live vaccine virus because ofincreased concentration of dissolved salts as a result of desiccation ofthe (tap) water particles, small amounts of a protein protectant, suchas skimmed milk, skimmed milk powder or gelatin can be added to aqueousphase.

In the spray or aerosol administration, generally a known amount of thevirus is discharged into a fixed air space rather than beingadministered directly to each broiler. Typically, for 20 broilersoccupying 1 m² of floor space, the average volume of air is about 0.14m³ or less per bird, depending upon the height of the ceiling. However,this type of administration is also possible in houses with open sides.In closed houses with mechanical ventilation, the ventilation is usuallyswitched off for a short period (e.g. 20 minutes). In such a case,vaccination is preferably carried out during periods when air conditionsare calm, in order to prevent the generated droplets to diffuse rapidlyout of the building.

For the generation of the small particles, conventional spray-apparatusand aerosol generators can be used. Also the drinking water vaccinationcan be carried out using a conventional apparatus. Details concerningconventional spray/aerosol—and drinking water vaccination can be foundin the “Compendium, administration of poultry vaccins” issued by theGezondheidsdienst voor Pluimvee, Doorn, the Netherlands, van Eck et al.,VI-VII, 1988.

In the administration by the drinking water route it is customary todeprive the broilers of water for about 2 to 4 hours before placing thevaccine containing water in front of them, and it is important thatthere is enough drinker space for all birds to drink evenly.

In order to prevent a dramatic reduction of the viable vaccine virus bythe presence of small amounts of chlorine, iron, zinc or copper ions inthe drinking water, preferably a protectant such as skim milk (powder)is added to the water containing vaccine.

The vaccine is diluted according to the age of the broilers so that anadequate amount of vaccine is allowed. The quantity of water generallyrequired per bird for the drinking water vaccination is about 10-15 mlfor 10 to 14 day old broilers and 20-30 ml for 3 to 8 weeks oldbroilers.

Live attenuated CAA to be used in the vaccine according to the inventionare known in the art. For example, Bülow von, V. and Fuchs, B. (J. Vet.Med. 33, 568-573, 1986) demonstrated that the pathogenicity of CAAstrain Cux-1 was decreased after 12 serial passages in MDCC-MSB1 cells.

Preferably, the vaccine according to the invention is derived from CAAwhich have been attenuated by serial passages of a virulent parentalstrain in embryonated eggs. Such CAA have the characteristic that theyinduce lesions in chicken embryo's but display a reduced pathogenicityfor young chicks, in particular for one-day-old chicks (European patentapplication No. 0533294).

A vaccine according to the invention can be prepared from any CAA strainavailable or obtainable from chickens suffering from infection with thispathogen. A number of CAA isolates have been described already in theprior art, e.g. the Cux-1 strain, the Gifu-1 strain, the TK-5803 strain,the CAA82-2 strain and strain 26P4 (CNCM accession No. I-1141).

The method for the preparation of the vaccine according to the presentinvention is conventionally and may include the steps of inoculating asusceptible substrate with attenuated CAA, in particular with such CAAwhich have been attenuated in embryonated eggs, propagating the CAA andharvesting CAA containing material.

Preferably, the substrate on which CAA is propagated are SPF embryonatedeggs. Embryonated eggs can be inoculated with for example 0.2 ml CAAcontaining suspension or homogenate comprising at least 10^(3.0) TCID₅₀per egg. Preferably, eggs are inoculated with at least 10^(4.5) TCID₅₀and subsequently incubated at 100° F. for 13 days. After 13 days the CAAproduct can be harvested by collecting the embryo's and/or the membranesand/or the allantoic fluid and appropriate homogenizing this material.The homogenate can be centrifuged thereafter for 10 min. at 2500 gfollowed by filtering the supernatant through a filter (100 μm).

Alternatively, the attenuated CAA can be inoculated onto a susceptiblecell culture, e.g. MDCC-MSB1 cells, followed by cultivation of the cellsand collecting the propagated virus.

Harvestable virus titres, preferably of at least about 10^(6.0)TCID₅₀/ml as assayed in MDCC-MSB1 cells can be obtained after 10-18 dayspost-inoculation, preferably 13 days after inoculation of embryonatedeggs.

The harvested fluids or virus material can be combined with apharmaceutically acceptable carrier or diluent as described below forfinal product filling and/or frozen in bulk or freeze-dried.

The vaccine according to the invention comprises an effective dosage ofthe live attenuated CAA, i.e. an amount of immunizing CAA that willinduce immunity in broilers against pathogen associated performancedrop. Immunity is defined herein as the induction of a significanthigher level performance results in broilers in a flock aftervaccination compared to an unvaccinated flock.

A vaccine according to the invention generally may comprise10^(2.0)-10^(6.0) TCID₅₀ of the live attenuated CAA per field dose foreach broiler, preferably 10^(4.0)-10^(5.0) TCID₅₀ of the virus per fielddose for each broiler.

The vaccine according to the invention containing the live virus can beprepared and marketed in the form of a suspension or in a lyophilizedform and additionally contains a pharmaceutically acceptable carrier ordiluent customary used for such compositions. Carriers includestabilizers, preservatives and buffers. Suitable stabilizers are, forexample SPGA, carbohydrates (such as sorbitol, mannitol, starch,sucrose, dextran, glutamate or glucose), proteins (such as dried milkserum, albumin or casein) or degradation products thereof. Suitablebuffers are for example alkali metal phosphates. Suitable preservativesare thimerosal, merthiolate and gentamicin. Diluents include water,aqueous buffer (such as buffered saline) alcohols and polyols (such asglycerol).

As the newly observed health problems in broilers resulting in aperformance drop emerges in the broilers from about 3-5 weeks of age,the vaccine should be administered to the broilers before or during theonset of the disease.

Preferably, the vaccine is administered to the broilers during the first4 weeks after hatch. In particular, the vaccine according to theinvention is effective if it is administered to broilers which arematernally immune, i.e. contain detectable levels (VN or Elisa titers≧1:32) of maternally derived antibodies (MDA) in their serum. It isdemonstrated herein that surprisingly the vaccine according to theinvention is able to induce a beneficial effect in the face of MDA andto raise a quick immune response such that immunity is achieved beforethe onset of the newly described health problems in broilers (3-5 weeksafter hatch).

EXAMPLE

Material and Methods

20 potential trial farms were identified by the local practitioner incooperation with the Regional Animal Health Service.

The broiler farm belonged to two different integrations.

The flocks sizes varied from 16,000 to 88,000 birds per round.

Approximately 700,000 birds were vaccinated, whereas approx. 260,000were left unvaccinated.

Various breeds were involved.

Vaccines/Vaccination-scheme

Participating flocks were vaccinated according to the following scheme:

Day old: ¼ dose ND Vaccine Nobilis Clone-30®, hatchery coarse spray.

Approx. 16 days: CAA, drinking water or spray.

Approx. 19 days: Gumboro Vaccine Nobilis D78®, drinking water, fulldose.

Approx. 21 days: IB+ND Vaccine Nobilis Ma5 Clone-30®, coarse spray, fulldose.

For aerosol CAA broiler vaccination per 10.000 doses (about 10^(5.0)TCID₅₀/animal), 1 liter water is used for reconstitution. The CAAvaccine is derived from the attenuated 19th egg-passaged 26P4 strain(European patent application No. 0533294). By means of an automizer(Atomist) an aerosol cloud is blown over the heads of the broilerchicks. By shutting down the ventilation (for 30 min.) the vaccine cloudis prevented from leaving the house. For the drinking wateradministration, a pre-solution of the required amount of the vaccinevirus (1.000 doses per 1000 chicks, about 10^(5.0) TCID₅₀/animal)mentioned-above in cold tap-water is prepared first. This solution wassubsequently added to the central drinking water supply, containing thatamount of water the group of broilers can consume within about 2 hours.After thirsting the broilers for about 2 hours the birds are admitted tothe central drinking nipple system. Of the first seven flocksvaccinated, 9 houses were vaccinated by Atomist (aerosol) and fourhouses were vaccinated by drinking water. As it was clear then thatthere was no difference in effects between the administration by Atomistand by the drinking water method, all subsequent vaccinated flocks werevaccinated by the aerosol method. On 11 production rounds, some houseswere left unvaccinated with CAA, as negative controls.

Parameters Used

Flocks were observed clinically for vaccination reaction.

Blood samples were taken at age of vaccination and at slaughter age, forserology.

Production data were collected through the integrations.

Production data were compared with production data of the previous 5rounds.

Where possible, production data of vaccinated houses were compared withdata of unvaccinated controls.

RESULTS

Vaccination Reaction

In none of the 20 flocks any adverse reaction was noticed. The mortalityfigures do not suggest any negative effect either, on the contrary (see“mortality”). There is a tendency towards lower mortality in thevaccinated flocks, which is strongest in the post vaccination mortality.

Serological Response

CAA-titers at time of vaccination (approx. 2 weeks of age): On 4 out of20 farms titers were below 4.5. On 16 out of 20 farms titers were above5.0 (maternal immunity).

Of 16 farms CAA-titers at slaughter age are available (approx. 6 weeks):On 3 out of 16 farms titers were found above 5.0. On 13 out of 16 farmsno titers above 4.0 were found, as serological indication of eitherfield infection or seroconversion after vaccination. This means that thevaccination has taken, without inducing seroconversion before slaughterage. If it is assumed that CAA was present on the farms and causingproblems, it means that the vaccine has prevented the field virus frominducing seroconversion. The seroconversion in some of the flocks mayhave been caused by CAA-field infection before the day of vaccination.

Production Data

Production index: The production index (PI) is calculated from theformula (Voeten and Brus):${PI} = \frac{\% \quad {surviving}\quad {birds}\quad \times \quad {kg}\quad {{{growth}/{day}}/{chicken}}\quad \times \quad 100}{{feed}\quad {conversion}}$

The formula takes in account mortality, growth and feed conversion, andcan be used to make comparisons between technical performances ofdifferent flocks at different times.

Results are shown in table 1.

TABLE 1 PI PI PI previously previously vaccinated (1 round) (5 rounds)Average 239 212 216 20 farms

The average PI of the 20 CAA-vaccinated broiler flocks was 239.

The average PI of the last previous non-vaccinated rounds on those 20flocks was 212, so there was an average improvement of 27 points.

The average PI of the previous 5 unvaccinated rounds on those farmsaveraged over the 20 flocks, was 216, so compared with that, thevaccinates scored 23 points better.

Of 16 out of the 20 flocks, the PI was the best performance, compared tothe 5 previous unvaccinated rounds.

Feed Conversion (FC)

Results are shown in table 2.

TABLE 2 FC 1500 g FC 1500 g FC 1500 g previously previously vaccinated(1 round) (5 rounds) Average 1.66 1.81 1.70 20 farms

The average FC (at a weight of the chicken of 1500 g) of the 20 flockswas 1.66.

The average FC of the last unvaccinated previous rounds on those farmswas 1.81. So there had been an average improvement of 0.15.

The average FC of the previous 5 unvaccinated rounds, averaged over the20 flocks, was 1.79. Compared to that figure, the trial rounds score0.13 points better.

Of 16 out of the 20 flocks, this was the best performance, compared tothe 5 previous unvaccinated rounds.

Growth Per Day

Results are shown in table 3.

TABLE 3 growth/day growth/day growth/day previously previouslyvaccinated (1 round) (5 rounds) Average 44.6 42.8 42.8 20 farms

16 out of 20 flocks have a higher growth per day, 4 have a lower growthrate, compared with the last previous unvaccinated round.

17 flocks improved, compared to the average over the 5 previousunvaccinated rounds.

The average growth per day was 44.6 grams.

The average growth per day of the last previous unvaccinated rounds was42.8, an average improvement of 1.8 grams.

The average growth of the previous 5 unvaccinated rounds, averaged over20 flocks was 42.8.

Total Mortality

Results are shown in table 4.

TABLE 4 Mortality (%) Mortality (%) Mortality (%) previously previouslyvaccinated (1 round) (5 rounds) Average 4.2 5.2 4.4 20 farms

13 flocks out of 20 experienced lower mortality, compared with the lastprevious unvaccinated round.

12 flocks out of 20 experienced lower mortality, compared to the averagemortality over the previous last 5 unvaccinated rounds.

The average mortality was 4.2%.

The average mortality in the last previous unvaccinated rounds was 5.2%,an improvement of 1.0%.

The average mortality in the 5 previous unvaccinated rounds was 4.4.Compared with this figure, the trial rounds improved by 0.2%.

What is claimed is:
 1. A method for the protection of broilers from thesub-clinical effects caused by horizontally transmitted CAA, comprisingadministering a live attenuated CAA vaccine mucosally to the broilersduring the first four week after hatch.
 2. The method according to claim1, wherein the vaccine is administered by spray or aerosol, or drinkingwater.
 3. The method according to claim 1, wherein the vaccine isadministered to maternally immune broilers.
 4. The method according toclaim 1, wherein 10^(2.0)-10^(6.0) TCID₅₀ of the live attenuated vaccineis administered to a broiler.
 5. A method for preventing production dropsyndrome of broilers, comprising mucosally administering a liveattenuated chicken anemia agent (CAA) vaccine to the broilers during thefirst four weeks after hatch, whereby, as compared to unvaccinatedbroilers, the production index is increased, feed conversion isimproved, growth per day is higher, and mortality is lower.
 6. Themethod according to claim 5, wherein the vaccine is administered byspray or aerosol, or drinking water.
 7. The method according to claim 5,wherein the vaccine is administered to maternally immune broilers. 8.The method according to claim 5, wherein 10^(2.0)-10^(6.0) TCID₅₀ of thelive attenuated vaccine is administered to a broiler.